Senthil K. Venugopal

Liver fibrosis causes downregulation of miRNA-150 and miRNA-194 in hepatic stellate cells, and their overexpression causes decreased stellate cell activation

Activation of hepatic stellate cells (HSC) results in their proliferation and in the secretion of extracellular matrix (ECM) proteins, which leads to hepatic fibrosis. microRNAs (miRNAs) have been shown to regulate various cell functions, such as proliferation, differentiation, and apoptosis. Hence, we have analyzed the miRNAs that were differentially expressed in HSC isolated from sham-operated and bile duct-ligated rats. Expression of two miRNAs, miRNA-150 and miRNA-194, was reduced in HSC isolated from fibrotic rats compared with sham-operated animals. These two miRNAs were overexpressed in LX-2 cells, and their ability to inhibit cell proliferation, the expression of smooth muscle alpha-actin (SMA), a marker for activation, and collagen type I, a marker for ECM secretion, was determined. Overexpression of these two miRNAs resulted in a significant inhibition of proliferation (P < 0.05) and reduced SMA and collagen I levels compared with either untreated cells or nonspecific miRNA-expressing cells. Next, the protein targets of these two miRNAs were found using bioinformatics approaches. C-myb was found to be a target for miRNA-150, and rac 1 was found to be one of the targets for miRNA-194. Therefore, we studied the expression of these two proteins by overexpressing these two miRNAs in LX-2 cells and found that overexpression of miRNA-150 and miRNA-194 resulted in a significant inhibition of c-myb and rac 1 expression, respectively. We conclude that both miRNA-150 and miRNA-194 inhibit HSC activation and ECM production, at least in part, via inhibition of c-myb and rac 1 expression.

Effect of C-reactive protein on vascular cells: Evidence for a proinflammatory, proatherogenic role

Purpose of reviewC-reactive protein (CRP) is the prototypic downstream marker of inflammation. High levels of CRP predict future cardiovascular risk in apparently healthy men and women. Recent evidence from different cell types suggests that CRP is not only a risk marker but may also be a participant in atherogenesis. This review will focus on the effects of CRP on different cells involved in atherosclerosis. Recent findingsCRP is shown to induce matrix metalloproteinase-1 (MMP-1) expression through the FcγRII and extracellular signal-related kinase pathway in U937 cells. MMPs are implicated in plaque instability. A recent report shows that CRP does not induce tissue factor in human monocytes directly, disputing the previous concept that CRP induces tissue factor in monocytes. CRP is shown to upregulate interleukin-8 in human aortic endothelial cells via nuclear factor-κB. CRP promotes monocyte chemoattractant protein-1-mediated chemotaxis by upregulating CC-chemokine receptor 2 expression in human monocytes. Also CRP is shown to attenuate endothelial progenitor cell survival, differentiation, and function via inhibiting nitric oxide. Human CRP transgenic animal models show that CRP promotes atherothrombosis and increases plasminogen activator inhibitor-1. Also, the classic dogma that CRP is produced exclusively in liver is challenged by recent data on the extrahepatic production of CRP in different cells including atherosclerotic lesions. SummaryAll this recent evidence along with earlier reports support a role for CRP in atherosclerosis.

Hepatitis B virus induces cell proliferation via HBx-induced microRNA-21 in hepatocellular carcinoma by targeting Programmed Cell Death Protein4 (PDCD4) and Phosphatase and Tensin Homologue (PTEN)

Hepatitis B viral infection-induced hepatocellular carcinoma is one of the major problems in the developing countries. One of the HBV proteins, HBx, modulates the host cell machinery via several mechanisms. In this study we hypothesized that HBV enhances cell proliferation via HBx-induced microRNA-21 in hepatocellular carcinoma. HBx gene was over-expressed, and miRNA-21 expression and cell proliferation were measured in Huh 7 and Hep G2 cells. miRNA-21 was over-expressed in these cells, cell proliferation and the target proteins were analyzed. To confirm the role of miRNA-21 in HBx-induced proliferation, Hep G 2.2.1.5 cells (a cell line that expresses HBV stably) were used for miRNA-21 inhibition studies. HBx over-expression enhanced proliferation (3.7- and 4.5-fold increase; n = 3; p<0.01) and miRNA-21 expression (24- and 36-fold increase, normalized with 5S rRNA; p<0.001) in Huh 7 and Hep G2 cells respectively. HBx also resulted in the inhibition of miRNA-21 target proteins, PDCD4 and PTEN. miRNA-21 resulted in a significant increase in proliferation (2- and 2.3-fold increase over control cells; p<0.05 in Huh 7 and Hep G2 cells respectively) and decreased target proteins, PDCD4 and PTEN expression. Anti-miR-21 resulted in a significant decrease in proliferation (p<0.05) and increased miRNA-21 target protein expression. We conclude that HBV infection enhances cell proliferation, at least in part, via HBx-induced miRNA-21 expression during hepatocellular carcinoma progression.

Butyrate induces ROS-mediated apoptosis by modulating miR-22/SIRT-1 pathway in hepatic cancer cells

Butyrate is one of the short chain fatty acids, produced by the gut microbiota during anaerobic fermentation of dietary fibres. It has been shown that it can inhibit tumor progression via suppressing histone deacetylase and can induce apoptosis in cancer cells. However, the comprehensive pathway by which butyrate mediates apoptosis and growth arrest in cancer cells still remains unclear. In this study, the role of miR-22 in butyrate-mediated ROS release and induction of apoptosis was determined in hepatic cells. Intracellular expression of miR-22 was increased when the Huh 7 cells were incubated with sodium butyrate. Over-expression of miR-22 or addition of sodium butyrate inhibited SIRT-1 expression and enhanced the ROS production. Incubation of cells with anti-miR-22 reversed the effects of butyrate. Butyrate induced apoptosis via ROS production, cytochrome c release and activation of caspase-3, whereas addition of N-acetyl cysteine or anti-miR-22 reversed these butyrate-induced effects. Furthermore, sodium butyrate inhibited cell growth and proliferation, whereas anti-miR-22 inhibited these butyrate-mediated changes. The expression of PTEN and gsk-3 was found to be increased while p-akt and β-catenin expression was decreased significantly by butyrate. These data showed that butyrate modulated both apoptosis and proliferation via miR-22 expression in hepatic cells.

Overexpression of microRNA‐30a inhibits hepatitis B virus X protein‐induced autophagosome formation in hepatic cells

Hepatitis B virus (HBV) enters the host and survives by using several mechanisms. One of the ways that HBV survives and replicates in the host cells is by inducing autophagy. Previous reports have shown that microRNA (miRNA)‐30a inhibits autophagosome formation in cancer cells. Hence, we hypothesized that overexpression of miRNA‐30a could inhibit HBV‐induced autophagosome formation in hepatic cells. To study this, both HepG2 cells and HepG2.2.1.5 cells (HBV‐expressing stable cell line) were transfected with miRNA‐30a, and the cells were collected either for RNA isolation or protein isolation after 72 h of transfection. Beclin‐1 expression was significantly higher in untransfected HepG2.2.1.5 cells than in HepG2 cells. Western blots showed that miRNA‐30a overexpression resulted in a significant decrease in beclin‐1 expression (eight‐fold and four‐fold in HepG2 and HepG2.2.1.5 cells, respectively) and c‐myc expression, whereas the numbers of terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL)‐positive cells were increased. In contrast, overexpression of HBV X protein (HBx) in HepG2 cells resulted in the enhancement of beclin‐1 (six‐fold increase as compared with the empty vector‐transfected cells) and c‐myc expression, whereas the numbers of TUNEL‐positive cells were reduced. To confirm these findings, HBx and miRNA‐30a were coexpressed in HepG2 cells, and the results showed significant inhibition of autophagosome formation and beclin‐1 and c‐myc expression, whereas apoptosis increased. These data demonstrate that HBx induces autophagosome formation via beclin‐1 expression, whereas miRNA‐30a overexpression could successfully inhibit the beclin‐1 expression induced by HBx, thereby modulating autophagosome formation in hepatic cells.

Global micro RNA expression profiling in the liver biopsies of Hepatitis B Virus infected patients suggests specific miRNA signatures for viral persistence and hepatocellular injury.

Hepatitis B virus (HBV) can manipulate the microRNA (miRNA) regulatory networks in infected cells to create a permissive environment for viral replication, cellular injury, disease onset, and its progression. The aim of the present study was to understand the miRNA networks and their target genes in the liver of hepatitis B patients involved in HBV replication, liver injury, and liver fibrosis. We investigated differentially expressed miRNAs by microarray in liver biopsy samples from different stages of HBV infection and liver disease (immune‐tolerant [n = 8], acute viral hepatitis [n = 8], no fibrosis [n = 16], early [F1+F2, n = 19] or late [F3+F4, n = 14] fibrosis, and healthy controls [n = 7]). miRNA expression levels were analyzed by unsupervised principal component analysis and hierarchical clustering. Analysis of miRNA–mRNA regulatory networks identified 17 miRNAs and 18 target gene interactions with four distinct nodes, each representing a stage‐specific gene regulation during disease progression. The immune‐tolerant group showed elevated miR‐199a‐5p, miR‐221‐3p, and Let‐7a‐3p levels, which could target genes involved in innate immune response and viral replication. In the acute viral hepatitis group, miR‐125b‐5p and miR‐3613‐3p were up, whereas miR‐940 was down, which might affect cell proliferation through the signal transducer and activator of transcription 3 pathway. In early fibrosis, miR‐34b‐3p, miR‐1224‐3p, and miR‐1227‐3p were up, while miR‐499a‐5p was down, which together possibly mediate chronic inflammation. In advanced fibrosis, miR‐1, miR‐10b‐5p, miR‐96‐5p, miR‐133b, and miR‐671‐5p were up, while miR‐20b‐5p and miR‐455‐3p were down, possibly allowing chronic disease progression. Interestingly, only 8 of 17 liver‐specific miRNAs exhibited a similar expression pattern in patient sera. Conclusion: miRNA signatures identified in this study corroborate previous findings and provide fresh insight into the understanding of HBV‐associated liver diseases which may be helpful in developing early‐stage disease diagnostics and targeted therapeutics. (Hepatology 2018;67:1695‐1709)

Humic acid inhibits HBV-induced autophagosome formation and induces apoptosis in HBV-transfected Hep G2 cells.

Hepatitis B Virus (HBV) utilizes several mechanisms to survive in the host cells and one of the main pathways being autophagosome formation. Humic acid (HA), one of the major components of Mineral pitch, is an Ayurvedic medicinal food, commonly used by the people of the Himalayan regions of Nepal and India for various body ailments. We hypothesized that HA could induce cell death and inhibit HBV-induced autophagy in hepatic cells. Incubation of Hep G2.2.1.5 cells (HepG2 cells stably expressing HBV) with HA (100 μM) inhibited both cell proliferation and autophagosome formation significantly, while apoptosis induction was enhanced. Western blot results showed that HA incubation resulted in decreased levels of beclin-1, SIRT-1 and c-myc, while caspase-3 and β-catenin expression were up-regulated. Western blot results showed that HA significantly inhibited the expression of HBx (3-fold with 50 μM and 5-fold with 100 μM) compared to control cells. When HA was incubated with HBx-transfected Hep G2 cells, HBx-induced autophagosome formation and beclin-1 levels were decreased. These data showed that HA induced apoptosis and inhibited HBV-induced autophagosome formation and proliferation in hepatoma cells.

Triiodothyronine stimulates VEGF expression and secretion via steroids and HIF-1α in murine Leydig cells

ABSTRACT Leydig cells are the principal steroidogenic cells of the testis. Leydig cells also secrete a number of growth factors including vascular endothelial growth factor (VEGF) which has been shown to regulate both testicular steroidogenesis and spermatogenesis. The thyroid hormone, T3, is known to stimulate steroidogenesis in Leydig cells. T3 has also been shown to stimulate VEGF production in a variety of cell lines. However, studies regarding the effect of T3 on VEGF synthesis and secretion by the Leydig cells were lacking. Therefore, we investigated the effect of T3 on VEGF synthesis and secretion in a mouse Leydig tumour cell line, MLTC-1. The effect of T3 was compared with that of LH/cAMP and hypoxia, two known stimulators of Leydig cell functions. The cells were treated with T3, 8-Br-cAMP (a cAMP analogue), or CoCl2 (a hypoxia mimetic) and VEGF secreted in the cell supernatant was measured using ELISA. The mRNA levels of VEGF were measured by quantitative RT-PCR. In the MLTC-1 cells, T3, 8-Br-cAMP, and CoCl2 stimulated VEGF mRNA levels and the protein secretion. T3 also increased steroid secretion as well as HIF-1α protein levels, two well-established upstream regulators of VEGF. Inhibitors of steroidogenesis as well as HIF-1α resulted in inhibition of T3-stimulated VEGF secretion by the MLTC-1 cells. This suggested a mediatory role of steroids and HIF-1α protein in T3-stimulated VEGF secretion by MLTC-1 cells. The mediation by steroids and HIF-1α were independent of each other. Abbreviations: 8-Br-cAMP: 8-bromo – 3ʹ, 5ʹ cyclic adenosine monophosphate; CoCl2: cobalt chloride; HIF-1α: hypoxia inducible factor −1α; LH: luteinizing hormone; T3: 3, 5, 3ʹ-L-triiodothyronine; VEGF: vascular endothelial growth factor

Augmenter of liver regeneration: A key protein in liver regeneration and pathophysiology: ALR and liver pathophysiology

Liver is constantly exposed to pathogens, viruses, chemicals, and toxins, and several of them cause injury, leading to the loss of liver mass and sometimes resulting in cirrhosis and cancer. Under physiological conditions, liver can regenerate if the loss of cells is less than the proliferation of hepatocytes. If the loss is more than the proliferation, the radical treatment available is liver transplantation. Due to this reason, the search for an alternative therapeutic agent has been the focus of liver research. Liver regeneration is regulated by several growth factors; one of the key factors is augmenter of liver regeneration (ALR). Involvement of ALR has been reported in crucial processes such as oxidative phosphorylation, maintenance of mitochondria and mitochondrial biogenesis, and regulation of autophagy and cell proliferation. Augmenter of liver regeneration has been observed to be involved in liver regeneration by not only overcoming cell cycle inhibition but by maintaining the stem cell pool as well. These observations have created curiosity regarding the possible role of ALR in maintenance of liver health. Thus, this review brings a concise presentation of the work done in areas exploring the role of ALR in normal liver physiology and in liver health maintenance by fighting liver diseases, such as liver failure, non‐alcoholic fatty liver disease/non‐alcoholic steatohepatitis, viral infections, cirrhosis, and hepatocellular carcinoma.

Circulating microRNAs: Possible role as non-invasive diagnostic biomarkers in liver disease.

Liver is the central organ for metabolism and the hepatocytes metabolize several drugs, hepatotoxins, alcohol, etc. Continuous exposure of the hepatocytes to these toxins result in various chronic diseases, such as alcoholic liver disease, non-alcoholic fatty liver disease, viral hepatitis and hepatocellular carcinoma. Although several diagnostic methods, such as serum markers, liver biopsy or imaging studies are currently available, most of these are either invasive or detect the disease at advanced stages. Hence, there is a need for new molecular markers that can be used for early detection of the disease. MicroRNAs (miRNAs) are naturally occurring, 20-22 nucleotide long, non-coding RNA molecules that regulate the gene expression at post-transcriptional levels, thereby modulating various biological functions. Their expression is deregulated under pathological conditions, and recent studies showed that they are secreted and can be detected in various body fluids. Since the cellular changes occur at earlier stages of the disease, detecting miRNAs in the body fluids could make them as potential novel biomarkers. Albeit, the difficulties in standardization procedures, cost and availability should be addressed before using them in the clinical arena. This review highlights the possible role of secreted miRNAs to use as early non-invasive diagnostic markers for liver disease.